Circular Dichroism Spectroscopy

Circular Dichroism (CD) is observed when optically active matter absorbs
left and right hand circular polarized light slightly differently. It is measured with a CD spectropolarimeter, which is relatively expensive (~$70k). The
instrument needs to be able to measure accurately in the far UV at wavelengths down to
190-170 nm. In addition, the difference in left and right handed absorbance A(l)-
A(r) is very small (usually in the range of 0.0001) corresponding to an
ellipticity of a few 1/100th of a degree. The CD is a function of wavelength. CD spectra
for distinct types of secondary structure present in peptides, proteins and nucleic acids
are different. The analysis of CD spectra can therefore yield valuable information about
secondary structure of biological macromolecules. See reference [1] for a review.

Linear polarized light can be viewed as a superposition of opposite
circular polarized light of equal amplitude and phase. A projection of the combined
amplitudes perpendicular to the propagation direction thus yields a line (figure 1a). When
this light passes through an optically active sample with a different absorbance A
for the two components, the amplitude of the stronger absorbed component will smaller than
that of the less absorbed component. The consequence is that a projection of the resulting
amplitude now yields an ellipse instead of the usual line (draw on a sheet of paper and
check). Note that the polarization direction has not changed. The occurrence of ellipticity
is called Circular Dichroism - it is not the same as optical rotation.
Rotation of the polarization plane (or the axes of the dichroic ellipse) by a small angle a
occurs when the phases for the 2 circular components become different, which requires a
difference in the refractive index n. This effect is called circular
birefringence. It can be shown that when CD exists, optical rotation must
exist as well, and they are directly related by a Kronig-Kramers transformation (see
anomalous x-ray dispersion (scattering factors f ' and f '') for more on K-K). The change
of optical rotation with wavelength is called optical rotary dispersion, ORD.
For more details on the physics see reference [3].

Figure 1 (a) Linear polarized light can be viewed as a
superposition of opposite circular polarized light of equal amplitude and phase. (b):
different absorption of the left- and right hand polarized component leads to ellipticity
(CD) and optical rotation (OR). The actual effect is minute and using actual numbers the
ellipse would still resemble a line.

The most common instruments around are the currently produced JASCO,
click here for JASCO U.K. home
page), Jobin Yvon,
OLIS, and
AVIV models, and the legendary,
out-of-production Cary 60 (with
the 6001 CD kit) or Cary 61. We have a Jasco 715 model with a temperature controller
and sample changer for rapid denaturation studies. The air cooled 150W Xenon lamp does not
necessitate water cooling, and the whole optics design and the piezo-mechanic modulator
are a great advantage over the old, floor-space space hogging Cary with its
Pockels-cells. You still need to
purge with ample nitrogen to get to lower wavelengths (below 190 nm). Meet JASCOMAN.

Most of the Carys are upgraded in some way. We had a great running
computerized Cary 60/6001 that is now donated to the UC Davis
student instrumentation lab, but somehow I think they really hate me for that.

Additives, buffers and stabilizing compounds: Any compound which absorbs in the region of interest (250 - 190 nm) should be
avoided. A buffer or detergent or other chemical should not be used unless it can be shown
that the compound in question will not mask the protein signal. For instance imidazole
cannot be used below 220 nm because it overwhelms the spectrum from then on. Therefore
ensure that only the minimum concentration of additives are present in the protein
solution.

Protein solution: From the above
follows that the protein solution should contain only those chemicals necessary to
maintain protein stability, and at the lowest concentrations possible. Avoid any chemical that is unnecessary for protein
stability/solubility. The protein itself should be as pure as possible, any additional
protein or peptide will contribute to the CD signal.

One may find that the protein concentration needs to be adjusted to
produce the best data. Changing this has a profound effect on the data, so small
increments or decrements are called for. If that does not produce reasonably good data, a
change in buffer composition may be necessary. It would also be a good idea to check the
sample for unforeseen aggregation via Dynamic Light Scattering (DNA repair enzymes are an
especially good example of this behavior). If absorption poses a problem, cells with
shorter path (0.1 mm) and a correspondingly increased protein concentration and longer
scan time can help.

As mentioned in the introduction, the difference in absorption to be
measured is very small. The differential absorption is usually a few 1/100ths to a few
1/10th of a percent, but it can be determined quite accurately. The raw data plotted on
the chart recorder represent the ellipticity of the sample in radians

which can be easily converted into degrees

To be able to compare these ellipticity values we need to convert into a normalized
value. The unit most commonly used in protein and peptide work is the mean molar
ellipticity per residue. We need to consider path length l, concentration c
, molecular weight M and number of residues

in proper units (CD spectroscopists use decimol)

which finally reduces to

The values for mean molar ellipticity per residue are usually in the 10.000's

Fitting of CD spectra

After baseline subtraction we are ready to analyze the data. As we mentioned already,
each of the three basic secondary structures of a polypeptide chain (helix, sheet, coil)
show a characteristic CD spectrum. A protein consisting of these elements should therefore
display a spectrum that can be deconvoluted into the tree individual contributions. This
has been realized quite early after CD was introduced and the standard curves shown to the
right were published in 1969 by Greenfield and Fasman [2]. Although those are actually for
poly-lysine only in different conformations, only little improvement in the accuracy of
fits has been achieved by attempting to generate other standard data sets from protein
spectra of known structure [5]. There are many limitations inherent in the method (such as
the lack of consideration of chromophore interaction between different structural regions
and neglect of other elements, 3-10 helices etc.), and the accuracy is not very high. I
personally think that a fit with an R-value below 10% is actually quite acceptable and
what one would expect for a qualitative assessment of protein folding. The method is,
however, very reliable for monitoring changes in the conformation of
proteins under different conditions (denaturation studies, unfolding experiments etc,
helix induction by TFE [4], see poly-glutamine example). Certain
backbone conformations can reveal quite different spectra - click highlight for an example
of an extended sheet of poly-Q.

Actual fit of a sample data against the Fasman poly-lys
standards (left) using my program CDFIT. The R-value of the fit is 6%, with a total helix
content of 80% and 20% random coil. The actual value is 77% total helix content. Note that
the results can be different depending on the region of fit [1] - a clear indication that
such fits must be treated with care.

Circular dichroism spectroscopy is used to gain information about the
secondary structure of proteins and polypeptides in solution.Benefits : Uses very little sample (200ul of 0.5 mg/ml solution in
standard cells), non-destructive. Relative changes due to influence of environment on
sample (pH, denaturants, temperature etc.) can be monitored very accurately.Drawbacks : interference with solvent absorption in the UV region, only
very dilute, non-absorbing buffers allow measurements below 200 nm. Absolute measurements
subject to a number of experimental errors, average accuracy of fits about +/- 10%. A CD
spectropolarimeter is relatively expensive.